Herpes Simplex Virus type 1 (HSV-1) protease must be catalytically active for successful nucleocapsid maturation, replication of the virus and infectivity of the virus. The proteases of HSV-1 and related herpes-group viruses are synthesized as precursor proteins (designated Pra) which undergo autoproteolytic processing during viral assembly to release a protease catalytic domain localized in the N-terminus of the precursor. In the case of HSV, the catalytic domain is within the N-terminal 247 amino acids of the 635 amino acid precursor protein. The mature processed form of the protease is a 27 kDa protein.
The only known protein substrates for the HSV protease are the viral protease precursor and the viral assembly protein known as ICP35 (Infected Cell Protein 35 or VP22a). Both ICP35 and the protease precursor are encoded by the U.sub.L 26 gene of HSV-1, with initiation of ICP35 translation occurring 306 codons after the initiation codon of the protease precursor, although the frequency in translation of ICP35 is approximately 10 times that of the precursor protease. (Liu, F. and Roizman, B. 1991, J. Virol. 65: 5149-5156, and Liu, F. and Roizman, B. 1991, J. Virol. 65: 206-212.) The reading frames for both proteins are the same, with the sequence of ICP35 identical to the C-terminus of the precursor protein.
The more abundant ICP35 is present in an immature form of the HSV capsids, known as B capsids, during capsid assembly within infected cell nuclei. The proteolytic conversion of ICP35 from the form found within the B capsids (ICP35 cd) to the shorter form found only within cell nuclei (ICP35 ef) is temporally linked with the conversion of B capsids to C capsids, which contain DNA. Thus, HSV protease action occurs within the cell nucleus and possibly within the viral capsid itself.
The mature form of the HSV-1 protease has been isolated following expression in E. coli and baculovirus systems. Inactivation with diisopropyl fluorophosphate was used to suggest the enzyme is a serine protease, but primary structure homology analyses have not revealed close relationships with well characterized groups of serine (or other) proteases. Peptides that represent cleavage sites within the natural protein substrates are cleaved at the bond between the characteristic Ala-Ser sequence found in protein substrates.
The k.sub.cat /K.sub.M of the purified mature HSV protease was found to be only 38 M.sup.-1 s.sup.-1 using peptide substrates, and a k.sub.cat /K.sub.M value of 36 M.sup.-1 s.sup.-1 has been reported using a fusion form of the enzyme with a similar assay condition, which includes 25% glycerol. These values are much lower than observed for other viral proteases such as rhinovirus 3C protease (1440 M.sup.-1 s.sup.-1) and HIV protease (13,000 M.sup.-1 s.sup.-1), and many orders of magnitude lower than other serine proteases such as chymotrypsin and thrombin (10.sup.7 M.sup.-1 s.sup.-1). While it is conceivable that the comparatively low catalytic efficiency of the HSV protease observed in vitro may be sufficient to account for its essential physiological role in nucleocapsid assembly, the low activity prompts consideration that some factor might enhance catalytic efficiency. In any event it would be desirable to be able to have a more active form of the protease in order to better study its activity.